DESCRIPTION Abstract: Epigenetics represents an exciting new frontier of biomedical research. A central yet essentially unexplored question in epigenetics is how epigenetic regulators are directed to specific loci in the genome to exert their function. It is known that a few transcriptional factors can recruit certain epigenetic factors to the promoters of target genes. However, this mechanism so far can only account for a handful of genes, and a tiny fraction of the genome. The latest Drosophila work in my lab suggest that PIWI-interacting RNAs (piRNAs) play a major and direct role in guiding epigenetic factors to many sites in the genome. Particularly, piRNA form complexes with PIWI proteins and directly bind to piRNA-complementary sites in the genome to regulate their epigenetic state. Furthermore, we have shown that PIWI directly recruits a key epigenetic factor called Heterochromatin Protein 1a to these sites. Based on these results, we propose a Piwi-piRNA guidance hypothesis, in which the Piwi-piRNA complex serves as a sequence- recognition machinery that recruits epigenetic effectors to specific genomic sites to execute epigenetic regulation. Here we propose to use combined genetic and genomic approaches to systematically test this hypothesis and to determine the contribution of the PIWI-piRNA- mediated mechanism to the genome at 10-basepair resolution. Specifically, we propose to: (1) Create the first functional epigenome map to determine the specific epigenetic effect of PIWI towards different regions of the genome; (2) develop a genome-wide epigenetic assay to examine the sufficiency and direct role of PIWI in regulating the transcriptional state of chromatin; and (3) take a four-pronged approach to directly test the role of piRNAs in guiding epigenetic factors to their sites. The proposed study should reveal a fascinating dimension of epigenetic regulation and generate a potentially transformative impact to the broad fields of epigenetics, genetics, molecular, developmental, and stem cell biology.